Project Summary Highly active antiretroviral therapy (HAART) has dramatically changed the prognosis for individuals infected with HIV-1. Yet, even when HIV-1 viremia has been well controlled by these drugs for years, termination of HAART results in viral rebound, most likely coming from latent provirus in long-lived memory CD4+ T cells. Most efforts to eradicate latent HIV-1 proviruses have focused on reactivation of proviral transcription to potentiate the elimination of reservoir cells harboring HIV-1 provirus, but these efforts have largely been unsuccessful. Alternative approaches for proviral elimination are therefore needed. Technological advances in gene editing tools provide a potential method for direct inactivation of latent HIV-1 provirus within reservoir cells. Specifically, the Cas9/CRISPR programmable nuclease system, a versatile platform for the generation of targeted double-strand breaks within the genome, has been shown to excise HIV-1 provirus in cell lines. However, the activity and precision of the Cas9/CRISPR system is suboptimal for clinical application. We have developed a novel nuclease architecture that combines the favorable cleavage activity of Cas9 with the targeting specificity of Transcription Activator-Like Effector (TALE) domains. This Cas9-TALE system dramatically improves the activity and precision of DNA cleavage. We propose to optimize this nuclease platform for the inactivation of HIV-1 provirus in memory CD4+ T cells, one of the primary cellular reservoirs of latent provirus. Development of a robust nuclease system for the efficient neutralization of provirus will also require a detailed map of the local chromatin landscape of the provirus in quiescent reservoir cells to identify areas at which it is particularly vulnerable to attack. However, obtaining the quantities of latently-infected primary cells needed for these analyses is not feasible. Consequently, in Aim 1 we will employ genome-editing technologies to generate populations of CD34+ cells containing a proviral insertion at a specific site and orientation within the genome. Once engrafted in NSG-BLT mice, these CD34+ cells will generate populations of resting CD4+ cells with uniform proviral integration sites for i-depth characterization, which may reveal important and unexpected drivers of latency in reservoir cells. In Aim 2, we will optimize the Cas9-TALE system to efficiently and precisely inactivate HIV-1 provirus with a high degree of precision. Initial optimization will be performed i Jurkat-based cell lines and then will move to an in vitro model of latently-infected central memory CD4+ cells. In Aim 3, these optimized nucleases will be evaluated on latent HIV provirus in a HAART-suppressed humanized mouse model of HIV-1 infection, and on resting CD4+ cells from patients undergoing HAART. Validated nucleases from our studies will be able to inactivate provirus efficiently from quiescent cells without compromising the host genome.